Viiatm7 rt qpcr system

Total RNA was extracted with a TRIzol reagent (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instructions. Amplification was performed using SYBR Green All-in-one TM qPCR Mix (GeneCopoeia) on a ViiA^TM7 RT-qPCR system (Applied Biosystems). The following thermocycling protocol was used: 95°C for 10 min, 40 cycles of 15 s at 95°C, 50 s at 60°C, and melting was done at 60°C. The primers for IL-1β, TNF-α, and β-actin were designed with Premier 5.0 software for rats. The sequences of the primers are shown in Table 2. Melting curves of all the samples were generated as controls for specificity. The expression data were normalized to the expression of β-actin with the 2^−ΔΔCt method.

TRIzol reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA and amplify cDNA according to the manufacturer’s instructions with Prime Script Kit (TaKaRa Bio Inc., Otsu, Japan) (1 ug per sample). RT-qPCR with three replicates was performed using SYBR green fluorescence assay (638320, TaKaRa Bio Inc.) on the ViiATM7 RT-qPCR system (Applied Biosystems, Carlsbad, CA). The primers for RT-qPCR were as follows: FLNB forward: 5′-ACTGTCATGGCCACAGATGG-3′; reverse: 5′-AAATCCCAGGCCGTTCATGT-3′; GAPDH forward: 5′-CGGAGTCAACGGATTTGGTCGTAT-3′; reverse: 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′; hsa_circ_0066351 forward: 5′-GAGGCCGATGTCATTGAGAA-3’; reverse: 5’-GCTCCACCAAGCCATGACAG-3’; Relative mRNA expression levels were calculated by the 2−(ΔΔCt) method and were normalized to the internal control (GAPDH), ΔCt = Ct (targeting gene) − Ct (GAPDH), and ΔΔCt =ΔCt (treated) – ΔCt (control).